The human immunodeficiency virus HIV is the causative agent of acquired immunodeficiency syndrome (AIDS), a disease characterised by the destruction of the immune system, particularly of the CD4+ T-cell, with attendant susceptibility to opportunistic infections. HIV infection is also associated with a precursor AIDs-related complex (ARC), a syndrome characterised by symptoms such as persistent generalised lymphadenopathy, fever and weight loss.
In common with other retroviruses, the HIV genome encodes protein precursors known as gag and gag-pol which are processed by the viral protease to afford the protease, reverse transcriptase (RT), endonuclease/integrase and mature structural proteins of the virus core. Interruption of this processing prevents the production of normally infectious virus. Considerable efforts have been directed towards the control of HIV by inhibition of virally encoded enzymes. In particular, much effort has been directed towards the inhibition of HIV protease and the HIV protease inhibitors (PIs) saquinavir, ritonavir, nelfinavir, indinavir, amprenavir and lopinavir have been approved for treatment of HIV infections. Because of the emergence of resistant virus during monotherapy, current clinical practice is to use such protease inhibitors in combination therapy, typically with RT inhibitors.
The emergence of resistant virus can be attributed to errors introduced by the HIV reverse transcriptase, in conjunction with a high virus replication rate. It is likely that mutations that lead to resistant virus occur spontaneously but remain undetectable until initiation of therapy leads to a selective pressure for the emergence of virus with replicative advantage over the wildtype population. In the context of HIV protease inhibition, accumulation of mutations that lead to a reduction in inhibitor binding while maintaining sufficient substrate turnover can lead to drug resistance. Although the onset of drug resistance can be delayed to some extent by the use of combinations of drugs, there remains a need for more effective HIV protease inhibitors that retain activity against PI-resistant and multi-PI resistant viruses.
This invention is concerned with novel HIV protease inhibitors or prodrugs thereof, a process for their manufacture, pharmaceutical compositions and the use of such compounds in medicine. In particular, the compounds are hydroxyethylamine tripeptide mimetics which act as inhibitors of the HIV aspartyl protease, an essential enzyme in the replicative life cycle of HIV. Consequently, the compounds of this invention may be advantageously used in the treatment of HIV infection, either alone or in combination with other inhibitors of HIV viral replication or with pharmacoenhancers such as cytochrome P450 inhibitors. This object could be achieved with the novel compounds of the general formula I 
The present invention comprises novel compounds of general formula I 
wherein R1 is H, hydroxy or NHR2 
wherein R2 is H, alkyl, alkenyl, alkynyl, arylalkyl, heterocyclylalkyl, cycloalkyl alkyl carbonyl, cycloalkyl carbonyl, aryl carbonyl, heterocyclyl carbonyl, heterocyclyl alkyl carbonyl, aryl alkyl carbonyl, alkyl oxy carbonyl, aryl alkyl oxy carbonyl, heterocyclyl alkyl oxy carbonyl, aryl heterocyclyl sulfonyl, alkyl sulfonyl, aryl sulfonyl, heterocyclyl sulfonyl or a group of the formula 
xe2x80x83wherein X is O or S and
R7and R8 independently are H, alkyl, aryl, heterocyclyl, aryl alkyl, heterocyclyl alkyl or R7 and R8 together with the nitrogen atom to which they are attached form a saturated ring optionally containing a further hetero atom or a group 
xe2x80x83wherein when n=0, Y represents O or S and R10 is H, alkyl, aryl alkyl, heterocyclyl alkyl, aryl, heterocyclyl or when n=1, Y represents N, R9 is H or alkyl and R10 H, alkyl, aryl alkyl, heterocyclyl alkyl, aryl, heterocyclyl or R9 and R10 taken together with the heteroatom to which they are attached form a heterocycle, R11 and R12 independently are H or alkyl or R11 and R12 taken together with the carbon atom to which they are attached form a cycle, R3, R4 independently are alkyl or taken together with the carbon atom to which they are attached form a carbocycle, R5 is alkyl, aryl alkyl, heterocyclyl alkyl or R4 and R5 taken together with the carbon and sulfur atom to which they are attached form a heterocycle and R6 is alkyl, aryl alkyl, heterocyclyl alkyl, alkyl oxy alkyl, hydroxy alkyl, amino alkyl, fluoro alkyl and R13 is H or the residue of an inorganic or an organic ester and R15 is aryl and pharmaceutically acceptable salts thereof, with the proviso that, if R3, R4 and R5 are methyl, R6 is tert.-butyl, R13 is H and if R15 is phenyl R2 is not benzyl oxycarbonyl and not 2-quinoline carbonyl.
The term alkyl defines an optionally substituted straight or branched alkyl chain carrying 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.
The term alkenyl defines an optionally substituted straight or branched alkenyl chain carrying 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms.
The term alkynyl defines an optionally substituted straight or branched alkynyl chain carrying 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms.
Alkyl accordingly preferably stands for methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl and tert.-butyl.
Alkenyl accordingly preferably is vinyl, 1-propenyl, 2-propenyl, i-propenyl, and butenyl and its isomers.
Alkynyl accordingly preferably is ethynyl, propynyl and its isomers, and butynyl and its isomers.
Suitable substituents of alkyl, alkenyl or alkynyl can be selected from one or more of aryl, heterocyclyl, carboxy, cyano, alkoxy, cycloalkyl oxy, aryl oxy, heterocyclyl oxy, hydroxy, alkyl carbonyl, cycloalkyl carbonyl, aryl carbonyl, heterocyclyl carbonyl, alkoxy carbonyl, cycloalkyl oxy carbonyl, aryl oxy carbonyl, heterocyclyl oxy carbonyl, amino carbonyl, alkyl amino carbonyl, dialkyl amino carbonyl, cycloalkyl amino carbonyl, aryl amino carbonyl, heterocyclyl amino carbonyl, amino, alkyl amino, dialkyl amino, alkenyl amino, alkynyl amino, cycloalkyl amino, aryl amino, heterocyclyl amino, alkyl carbonyl amino, dialkyl carbonyl amino, cycloalkyl carbonyl amino, aryl carbonyl amino, heterocyclyl carbonyl amino, alkoxy carbonyl amino, cycloalkyl oxy carbonyl amino, aryloxy carbonyl amino, heterocylyl oxy carbonyl amino, alkyl amino carbonyl amino, dialkyl amino carbonyl amino, cycloalkyl amino carbonyl amino, aryl amino carbonyl amino, heterocyclyl amino carbonyl amino alkyl sulfonyl amino, cycloalkyl sulfonyl amino, aryl sulfonyl amino, heterocyclyl sulfonyl amino, nitro, alkyl sulfonyl, cycloalkyl sulfonyl, aryl sulfonyl, heterocyclyl sulfonyl, thio, alkyl thio, cycloalkyl thio, aryl thio, heterocyclyl thio or halogen.
In all cases above where there are NH groups, the free hydrogen may also be substituted, preferably with lower alkyl.
Cycloalkyl has the meaning of an optionally substituted cycloalkyl group containing 3 to 8 carbon atoms, preferably 3 to 6 carbon atoms e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl or adamantyl which can also be benz-fused to an optionally substituted saturated, partially unsaturated or aromatic monocyclic, bicyclic or tricyclic heterocycle or carbocycle, e.g. to phenyl.
The term aryl denotes optionally substituted phenyl and naphthyl, both optionally benz-fused to an optionally substituted saturated, partially unsaturated or aromatic monocyclic, bicyclic or tricyclic heterocycle or carbocycle e.g. to cyclohexyl or cyclopentyl.
The term heterocyclyl stands for an optionally substituted saturated, partially unsaturated or aromatic monocyclic, bicyclic or tricyclic heterocycle which contains one or more hetero atoms selected from nitrogen, oxygen and sulfur which can also be benz-fused to an optionally substituted saturated, partially unsaturated or aromatic monocyclic, bicyclic or tricyclic carbocycle or heterocycle.
Examples of suitable heterocycles are oxazolyl, isoxazolyl, furyl, tetrahydrofuryl, 1,3-dioxolanyl, dihydropyranyl, thienyl, pyrazinyl, isothiazolyl, isoquinolinyl, indolyl, indazolyl, quinolinyl, dihydrooxazolyl, pyrimidinyl, benzofuranyl, tetrazolyl, pyrrolidinonyl, (N-oxide)-pyridinyl, pyrrolyl, triazolyl e.g. 1,2,4-triazolyl, pyrazolyl, benzotriazolyl, piperidinyl, morpholinyl, thiazolyl, pyridinyl, dihydrothiazolyl, imidazolidinyl, pyrazolinyl, benzothienyl, piperazinyl, imidazolyl, thiadiazolyl e.g. 1,2,3-thiadiazolyl, and benzothiazolyl.
Suitable substituents for cycloalkyl, aryl, heterocyclyl can be selected from those named for alkyl, in addition, however, alkyl, alkenyl and alkynyl are substituents to be added to the selection.
The term halogen stands for fluorine, chlorine, bromine and iodine.
The term residue of an inorganic ester stands for a sulfate of the formula xe2x80x94SO2OH or a phosphate of the formula xe2x80x94PO(OH)2.
The term residue of an organic ester defines an acyl group as e.g. described in the European Patent Application EP A1 0 594 540 for group R1.
Suitable residues of an organic ester are defined in R13 as being a group of formula 
wherein R14 is alkyl, alkenyl, cycloalkyl, aryl, aryl alkyl heterocyclyl, a group xe2x80x94CH2 (CH2CH2O)mCH3, wherein m is an integer from 0 to 10, or a carbonyl group-linked radical of an aminoacid.
With the exception that the alkyl and the alkenyl chain can carry up to 20 carbon atoms the meaning of the terms alkyl, alkenyl, cycloalkyl, aryl, heterocyclyl is the same as outlined above.
The term xe2x80x9ccarbonyl group-linked radical of an aminoacidxe2x80x9d stands for a radical of a natural or unnatural amino acid selected from e.g. glycine, alanine, leucine, isoleucine, phenylalanine, lysine, methionine, threonine, tryptophan, valine, serine, glutamine, etc which is linked to the carbonyl group of 
Any functional (i.e. reactive) group present in a side-chain may be protected, with the protecting group being a group which is known per se, for example, as described in xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, 2nd Ed., T. W. Greene and P. G. M. Wuts, John Wiley and Sons, New York, N.Y., 1991. For example, an amino group can be protected by a tert-butoxycarbonyl (BOC), formyl, trityl, benzyloxycarbonyl (Z), 9-fluorenylmethyloxcarbonyl (FMOC), trifluoroacetyl, 2-(biphenylyl)isopropoxycarbonyl or isobornyloxycarbonyl group or in the form of a phthalimido group; or a hydroxyl group can be protected by a tert-butyldimethylsilyl, tetrahydropyranyl, 4-methoxybenzyl, or benzyl or acetate etc; or a carboxyl group can be protected in the form of an ester, for example as a methyl or benzyl or tert. butyl ester. The protecting group may be retained in the final compound or optionally removed by techniques known in the art.
The compounds of this invention are characterized by a core structure with fixed stereochemistry as shown in general formula
The residues R1 to R15 in compounds of this invention may contain one or more asymmetric carbon atoms and may therefore occur as single enantiomers, racemates and racemic mixtures, individual diastereomers and diastereomeric mixtures. Furthermore, where a compound of the invention contains an olefinic double bond, this can have the (E) or (Z) configuration. Also, each chiral centre may be of the R or S configuration. All such isomeric forms of these compounds are embraced by the present invention.
Compounds of formula (I) which are acidic can form pharmaceutically acceptable salts with bases such as alkali metal hydroxides, e.g. sodium hydroxide, potassium hydroxide and the like; alkaline earth metal hydroxides, e.g. calcium hydroxide, barium hydroxide, magnesium hydroxide and the like; with organic bases e.g. N-ethyl piperidine, dibenzylaminen and the like. Those compounds of formula (I) which are basic can form pharmaceutically acceptable salts with inorganic acids, e.g. hydrohalic acids such as hydrochloric acid and hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid and the like; and with organic acids, e.g. acetic acid, tartaric acid, succinic acid, fumaric acid, maleic acid, malic acid, salicylic acid, citric acid, methanesulphonic acid, p-toluene sulphonic acid and the like. The formation and isolation of such salts can be carried out according to methods known in the art.
Preferred compounds of formula (I) are those having the formula 
wherein R2, R3, R4, R5, R6, R13 and R15 are as above.
In a further preferred embodiment R3, R4 and R5 have the meaning of methyl, R6 has the meaning of tert-butyl or hydroxy tert-butyl and R15 has the meaning of phenyl.
In a further preferred embodiment R2 is alkyl carbonyl, cycloalkyl carbonyl, aryl carbonyl, heterocyclyl carbonyl, heterocyclyl alkyl carbonyl or a group of the formula 
wherein when n=0, Y represents O or S
and R10 alkyl, aryl alkyl, heterocyclyl alkyl, aryl, heterocyclyl or when n=1, Y represents N, R9 is H and R10 is alkyl, aryl alkyl, heterocyclyl alkyl, aryl, heterocyclyl
and wherein R11 and R12 independently are H.
Still further preferred are compounds wherein R3, R4 and R5 are methyl, R6 is tert-butyl, R15 is phenyl and R2 is alkyl carbonyl, cycloalkyl carbonyl, aryl carbonyl, heterocyclyl carbonyl, heterocyclyl alkyl carbonyl or a group of the formula 
wherein when n=0, Y represents O or S
and R10 is alkyl,aryl alkyl, heterocyclyl alkyl, aryl, heterocyclyl or when n=1, Y represents N, R9 is H and R10 is alkyl, aryl alkyl, heterocyclyl alkyl, aryl, heterocyclyl
and wherein R11 and R12 independently are H.
Still further preferred are compounds wherein R3, R4, R5 are methyl, R6 is tert-butyl, R15 is phenyl and R2 is aryl carbonyl, heterocyclyl carbonyl, or a group of the formula 
wherein Y represents O, NH, S, CH2 and R10 is aryl or heterocyclyl.
In a still further preferred embodiment R13 has the meaning of H.
Examples of compounds of formula I or II with the meaning of R13 is H are set out in table A below.
In another preferred embodiment R13 has the meaning of xe2x80x94SO2OH, xe2x80x94PO(OH)2 or of a group 
wherein R14 is alkyl, alkenyl, cycloalkyl, aryl, aryl alkyl, heterocyclyl, a group xe2x80x94CH2 (CH2CH2O)mCH3, wherein m is an integer from 0 to 10, or a carbonyl group-linked radical of an aminoacid.
Examples of compounds of formula I or II with R13 not being H are set out in Table B below.
Further preferred compounds of formula (I) are those having the formula 
wherein R3, R4, R5, R6, R13 and R15 are as above and R20 is heterocyclyl.
More preferred compounds of formula (X) are those where R3, R4 and R5 are methyl, R6 is tert-butyl, R13 is H and R15 is phenyl.
Examples of such preferred compounds of formula (X) are listed below.
N-tert-Butyl-1,2,3,4,4a(S),5,6,7,8,8a(S)-decahydro-2-[2(R)-hydroxy-3(S)-[[3-(methanesulfonyl)-N-[2-(3-pyridyloxy)acetyl]-L-valyl]amino]-4-phenylbutyl]-3(S)-isoquinolinecarboxamide
N-tert-Butyl-1,2,3,4,4a(S),5,6,7,8,8a(S)-decahydro-2-[2(R)-hydroxy-3(S)-[[3-(methanesulfonyl)-N-[2-(2-pyridyloxy)acetyl]-L-valyl]amino]-4-phenylbutyl]-3(S)-isoquinolinecarboxamide
N-tert-Butyl-1,2,3,4,4a(S),5,6,7,8,8a(S)-decahydro-2-[2(R)-hydroxy-3(S)-[[3-(methanesulfonyl)-N-[2-(6-methyl-3-pyridyloxy)acetyl]-L-valyl]amino]-4-phenylbutyl]-3(S)-isoquinolinecarboxamide
N-tert-Butyl-1,2,3,4,4a(S),5,6,7,8,8a(S)-decahydro-2-[2(R)-hydroxy-3(S)-[[3-(methanesulfonyl)-N-[2-(3-pyrazinyloxy)acetyl]-L-valyl]amino]-4-phenylbutyl]-3(S) -isoquinolinecarboxamide
2-(2-Hydroxy-3-{3-methanesulfonyl-3-methyl-2-[2-(pyrimidin-2-yloxy)-acetylamino]butyrylamino}-4-phenyl-butyl)-decahydro-isoquinoline-3-carboxylic acid tert-butylamide
Most preferred compound is
N-tert-Butyl-1,2,3,4,4a(S),5,6,7,8,8a(S)-decahydro-2-[2(R)-hydroxy-3(S)-[[3-(methanesulfonyl)-N-[2-(3-pyridyloxy)acetyl]-L-valyl]amino]-4-phenylbutyl]-3(S)-isoquinolinecarboxamide its pharmaceutically acceptable salts and esters.
The hydroxyethylamine compounds provided by the present invention are potent inhibitors, or prodrugs thereof, of the HIV aspartyl protease, an essential enzyme in the replicative cycle of the HIV virus. They accordingly are therapeutically active substances in the treatment of HIV-mediated diseases and therefore can be used as medicaments, either alone or combined with other therapeutically active agents.
The hydroxyethylamine compounds provided by the present invention are, in particular, useful in combating HIV disease states such as AIDS.
Compounds of the invention with formula I wherein R1 is NHR2 can be prepared from a compound of formula III 
wherein R3, R4, R5, R6 and R15 are as above.
For a compound of formula I wherein R1 is NHR2 in which R2 is alkyl carbonyl, cycloalkyl carbonyl, aryl carbonyl, heterocyclyl carbonyl, heterocyclyl alkyl carbonyl, aryl alkyl carbonyl, alkyl oxy carbonyl, aryl alkyl oxy carbonyl, heterocyclyl alkyl oxy carbonyl, sulfonyl, alkyl sulfonyl, aryl sulfonyl, heterocyclyl sulfonyl, or a group of formula 
wherein R9, R10, R11, and R12 are as above.
the compound of formula III is reacted with an appropriate acid derivative such as an acyl halide, mixed anhydride etc.
Alternatively when n=1, Y represents N, R9 is H and R10 is alkyl, aryl alkyl, heterocyclyl alkyl, the compound of formula III is reacted with N-protected glycine, deprotected and reacted with an aldehyde or ketone under reductive conditions as described in embodiment b) of the process
For a compound of formula I wherein R1 is NHR2 in which R2 is, alkyl, alkenyl, alkynyl, arylalkyl, heterocyclylalkyl, cycloalkyl, a compound of formula III is reacted with an aldehyde or ketone under reductive conditions.
For a compound of formula I wherein R1 is NHR2 in which R2 is a group of formula 
in which X, R7 and R8 have the same meaning as described previously, a compound of formula III is reacted with reagents described in the art for the formation of ureas and thioureas.
For a compound of formula I wherein R1 is NHR2 in which R2 is a heterocycle a compound of formula III is reacted according to methods described in the art for the formation of heterocycles. For example, in the case where R2 is thiazole, by using the Hansch synthesis according to Scheme 1, by converting a compound of formula III into the thiourea IV followed by reaction of IV with the required xcex1-haloketone or xcex1-haloaldehyde 
in said scheme, R2, R3, R4, R5, R6 and R15 are as previously described; R16 has the meaning of H. alkyl, alkoxy carbonyl, aryl, heterocyclyl; R17 has the meaning of H, alkyl, aryl, heterocyclyl; R18 is the same as R16; and R19 is the same as R17. Hal has the meaning of a halogen atom selected of, chlorine, bromine and iodine.
For a compound of formula I wherein R1 is NHR2 in which R2 is aryl, a compound of formula III is reacted with aryl halides under transition metal catalysed conditions known in the art. Alternatively the amino acid derived from deprotection of compounds V (Scheme 3) can be reacted with aryl halides under similar conditions prior to coupling with compounds of formula VI (Scheme 3).
For a compound of formula I in which R6 is alkyl, aryl alkyl, heterocyclyl alkyl, alkyl oxy alkyl, hydroxy alkyl,amino alkyl, fluoro alkyl, a compound of formula IVa (Scheme 2) is reacted with the appropriate amine under conditions of Lewis acid catalysis 
in said scheme R1, R3, R4, R5, R6 and R15 are as previously described.
For a compound of formula I in which R13 has the meaning of xe2x80x94SO2OH, xe2x80x94PO(OH)2, or a group 
wherein R14 is as previously described,
a compound of formula I in which R13 is H is reacted with the appropriate activated acid derivative or, in the case of an amino acid, an amino-protected form thereof, according to methods described in the art for the formation of esters.
In accordance with embodiment a) of the process, suitable reagents which yield alkyl carbonyl, cycloalkyl carbonyl, aryl carbonyl, heterocycly carbonyl, aryl alkyl carbonyl, heterocycly alkyl carbonyl, alkyl oxy carbonyl, aryl alkyl oxy carbonyl, heterocyclyl alkyl oxy carbonyl, sulfonyl, alkyl sulfonyl, aryl sulfonyl, or heterocyclyl sulfonyl amines or a group of formula 
wherein R9, R10, R11 and R12 are as previously described,
are the corresponding acids or reactive derivatives thereof, such as the corresponding acid halides (e.g. acid chlorides), acid anhydrides, mixed anhydrides, activated esters etc. The reaction of III with the aforementioned reagents is carried out in accordance with methods described in the art for example in text books on organic chemistry such as J. March (1992) xe2x80x9cAdvanced Organic Chemistry: Reactions, Mechanisms and Structurexe2x80x9d, 4th ed. John Wiley and Sons. Thus when an acid is used, the reaction is preferably carried out in the presence of condensation agents such as N-ethyl-Nxe2x80x2(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC.HCl) in the presence of hydroxybenzotriazole (HOBT). This reaction is conveniently carried out in an inert organic solvent such as tetrahydrofuran (THF), dichloromethane or dimethylformamide at a temperature from xe2x88x9210xc2x0 C. to +25xc2x0 C. When a reactive derivative is used the reaction can be carried out in an inert solvent such as dichloromethane or tetrahydrofuran in the presence of an organic base (e.g. N-ethylmorpholine, triethylamine etc) at a temperature from xe2x88x9210xc2x0 C. to 25xc2x0 C.
In accordance with embodiment b) of the process, reaction of compounds of formula III with an aldehyde or ketone can be carried out according to methods described in the art for the reductive amination of aldehydes and ketones. For example, text books on organic chemistry such as J. March (1992) xe2x80x9cAdvanced Organic Chemistry: Reactions, Mechanisms and Structurexe2x80x9d, 4th ed. John Wiley and Sons can be consulted. Thus, for example, the reaction is conveniently carried out with sodiumtriacetoxyborohydride in an inert halogenated solvent such as dichloroethane in the presence of acetic acid according to the method described by A. F. Abdel-Magid et al; Tetrahedron Letters 1990, 31, 5595.
In accordance with embodiment c) of the process, the reaction can be carried out according to methods known in the art, for example in text books on organic chemistry such as J. March (1992) xe2x80x9cAdvanced Organic Chemistry: Reactions, Mechanisms and Structurexe2x80x9d, 4th ed. John Wiley and Sons. Thus, for example, for a compound in which X is O, the reaction can be carried out by reaction of compounds of formula III with para-nitrophenylchloroformate in the presence of an inorganic base such as sodium hydrogen carbonate followed by reaction with an amine R7R8NH in the presence of an organic base such as triethylamine where R7 and R8 have the significance given earlier. (See for example N. Choy et al. Org. Prep. Proced. Int. 1996, 28(2), 173-7). The reaction is conveniently carried out in an inert polar solvent such as acetonitrile at a temperature between 0xc2x0 C. and 25xc2x0 C. When X is O or S and one of R7 or R8 is H the reaction can be conveniently carried out by the reaction of a compound of formula III with an isocyanate (R7Nxe2x95x90Cxe2x95x90O or R8Nxe2x95x90Cxe2x95x90O) or isothiocyanate (R7Nxe2x95x90Cxe2x95x90S or R8Nxe2x95x90Cxe2x95x90S) according to methods described in the art.
In accordance with embodiment d) of the process, the reaction can be carried out according to methods described in text books on heterocyclic chemistry such as T. L. Gilchrist (1992) xe2x80x9cHeterocyclic Chemistryxe2x80x9d, 2nd ed. John Wiley and Sons. For example when R2 is thiazole, the reaction can be carried out by heating a mixture of compound IV and the xcex1-halocarbonyl compound in an appropriate solvent such as an alkanol (e.g. ethanol). Compound IV can be readily prepared from compound III according to known methods, for example by reaction with benzoyl isothiocyanate in refluxing acetone followed by hydrolysis with an inorganic base such as potassium carbonate in a mixture of a polar organic solvent and water. (See for example N. M. Olken et al J. Med. Chem. 1992, 35, 1137).
In accordance with embodiment e) of the process, the reaction of amino acids (derived from compounds of formula V by deprotection) with aryl halides, e.g. bromobenzene, can be carried out in the presence of copper salts, e.g copper iodide in dimethyl acetamide. See for example D. Ma et al, J. Amer. Chem. Soc 1998, 120, 12467.
In accordance with embodiment f) of the process, the reaction of compounds of formula IVa with amines R6NH2 is carried out using methods described in the art for example using a reagent derived from the amine and an aluminium derived Lewis acid, e.g. trimethylaluminium, at ambient temperature in an inert solvent such as dichloromethane or toluene. (See for example S. M. Weinreb et al, Tetrahedron Letters 1977, 4171)
In accordance with embodiment g) of the process, the reaction can be carried out according to methods known in the art for the formation of esters, see for example text books on organic chemistry such as J. March (1992) xe2x80x9cAdvanced Organic Chemistry: Reactions, Mechanisms and Structurexe2x80x9d, 4th ed. John Wiley and Sons. For example the reaction is conveniently carried out at ambient temperature using the carboxylic acid derivative and a peptide coupling reagent such as EDAC.HCl in an inert solvent such as dichloromethane in the presence of 4-dimethylaminopyridine as a catalyst. Alternatively the acyl halide can be used in an inert solvent in the presence of pyridine and 4-dimethylaminopyridine as a catalyst at a temperature between 0xc2x0 C. and 25xc2x0 C.
Compounds of formula III which are used as starting materials in embodiments a-e are either known as can be prepared according to Scheme 3. Thus reaction of a compound of formula V with a compound of formula VI can be carried out in accordance with methods known in peptide chemistry to give a compound of formula VII (see J. Jones (1994), xe2x80x9cThe Chemical Synthesis of Peptidesxe2x80x9d, Oxford University Press). The term xe2x80x9camino protecting groupxe2x80x9d (Prot) as used herein refers to groups employed in peptide chemistry such as a tert-butoxycarbonyl group (BOC) or a 9-fluorenylmethyloxycarbonyl group (FMOC). The preferred amino protecting group (Prot) for this reaction is a 9-fluorenylmethyloxycarbonyl group. This reaction is preferably carried out by reaction of a compound of formula V with a chloroformate (e.g. isobutylchloroformate) in the presence of an organic base such as N-ethylmorpholine to generate a mixed anhydride which is subsequently reacted with compounds of formula VI. The reaction is conveniently carried out in an inert solvent such as an ether (e.g. diethyl ether, tetrahydrofuran, etc) or an aliphatic halogenated solvent (e.g. dichloromethane) at a low temperature, suitably at about xe2x88x9210xc2x0 C. to 5xc2x0 C. Conversion of compounds of formula VII into compounds of formula III is carried out using known methods employed in peptide chemistry for the deprotection of the amino group of amino acids. For example when the amino protecting group is FMOC the reaction is conveniently conducted by reaction of compounds of formula VII with piperidine in dimethylformamide or dichloromethane at room temperature. 
in said scheme R3, R4, R5, R6 and R15 have the meaning previously described.
Compounds of formula V where R3 and R4 are methyl can be prepared from penicillamine according to Scheme 4. 
in said scheme R5 is as previously described.
Thus reaction of L-penicillamine with an alkyl halide R5X, where R5 has the significance given earlier and X is a halogen (e.g. bromide), in the presence of an inorganic base such as potassium carbonate, followed by reaction with a reagent for introducing amino acid protecting groups (e.g. FMOCONSu or BOC2O) gives compounds of formula VIII. The reaction can be carried out at room temperature in a mixed solvent system consisting of water and an organic solvent preferably dioxane. Compounds of formula VIII are oxidized to compounds of formula Va according to known procedures preferably by reaction with Oxone (K. S. Webb, Tetrahedron Lett. 1994, 35(21), 3457-60).
Other compounds of formula V can be prepared by analogous routes from penicillamine analogues described in the art.
Compounds of formula VI can be prepared according to the known procedures described in the art, for example EP 432695 A2.
Compounds of formula I in which R1 is hydroxy can be prepared according to methods described in the art, for example A. N. Cook et al; J. Chem. Soc. 1949, 1022. For example, deprotection of the amino acids V followed by diazotization, hydrolysis, and coupling to compounds of formula VI according to the methods described above gives compounds of formula I in which R1 is hydroxy.
Compounds of formula I in which R1 is H can be prepared from compounds of formula VI and compounds of formula IX (Scheme 5) in a manner analogous to that already described. Compounds of formula IX can be prepared from the appropriate acrylic acid and thiol according to methods decribed in the art and outlined in Scheme 5 (See for example G. Pattenden et al, J. Chem. Soc., Perkin Trans. 1, 1992, (10), 1215-21) 
in said scheme R3, R4 and R5 are as previously described.
The starting materials of formula V, VI, IX and their reactive derivatives, insofar as they are not known compounds or analogues of known compounds, can be prepared in a similar manner to the known compounds or as described in the examples hereinafter or by analogy thereto. Moreover, the reagents used in embodiments a-g are generally known compounds.
Reagents required for the introduction of groups of formula 
wherein R9, R10, R11 and R12 are as previously described are the corresponding carboxylic acids, or activated derivatives thereof, which themselves are known compounds or can be readily prepared by analogy to known compounds. For example when n=0, Y represents O or S and R10 is aryl or heterocyclyl the reagent is prepared by reaction of the appropriate alcohol (e.g. 3-hydroxypyridine) with tert-butylbromoacetate under basic conditions (e.g. sodium hydride in dimethylformamide or potassium carbonate in acetone) followed by acid-catalysed deprotection (e.g. hydrochloric acid in ether, hydrobromic acid in acetic acid or trifluoroacetic acid in dichloromethane). Similarly, when n=1 and Y represents N, the reagent can be prepared by similar methods in which the amine R9R10NH is used instead of the alcohol and without added base. Alternatively, glycine t-butyl ester can be reductively aminated with an aldehyde or ketone under analogous conditions to those described above in embodiment b) of the process, followed by acid catalysed deprotection of the carboxylic acid group; preferably with HBr in acetic acid.
HIV protease inhibitory activity was assessed using an adaptation of the method of Matayoshi. et al. [Matayoshi E. D. et al (1990). Science. 247. 954-958]
Crude HIV-1 protease was prepared from E. coli pPTxcex94N. Cultures were grown at 30xc2x0 in M9 medium supplemented with 0.2% casamino acids, 100 xcexcg/ml ampicillin and 25 xcexcg/ml thiamine until OD600=0.5-0.6, and the temperature was raised to 42xc2x0 to induce expression of the protease. After 1.5 hours, the cells were harvested and the pellets stored at xe2x88x9270xc2x0 until required.
The protease was prepared by lysis of the cells in a French pressure cell followed by precipitation of the enzyme with ammonium sulfate at 30% saturation.
The assay was based on intramolecular fluorescence energy transfer using a quenched fluorogenic substrate DABCYL-Ser.Gln.Asn.Tyr.Pro.Ile.Val.Gln.-EDANS, the peptide sequence of which was derived from one of the natural polypeptide processing sites of HIV-1 protease.
The peptide substrate was dissolved in spectroscopic grade dimethyl sulphoxide (DMSO) to give a stock solution of 500 xcexcM. Inhibitors were dissolved in a 1:9 mixture of DMSO:0.1% aqueous Tween 20 to give inhibitor concentrations 20xc3x97 more concentrated than the desired final concentration. The assay buffer comprised 0.1M sodium acetate pH 4.7, 8 mM EDTA, 0.2 M NaCl.
10 xcexcl HIV-1 protease diluted in a 1:1 mixture of 0.1% Tween:assay buffer (concentration adjusted to give approximately 20% substrate turnover) was added to a mixture comprising 455 xcexcl assay buffer, 25 xcexcl inhibitor solution, 10 xcexcl substrate solution.
Tubes were incubated for 2 hours at 37xc2x0 and the reaction was terminated by the addition of 500 xcexcl of a 2:1 mixture of DMSO:50 mM Tricine pH 8.5. Fluorescence was measured in a fluorescence spectrophotometer, excitation xcex=340 nm, emission xcex=492 nm.
Anti-HIV antiviral activity was assessed using an adaptation of the method of Pauwels et al. [Pauwels et al., 1988, J. Virol. Methods 20: 309-321]. The method is based on the ability of compounds to protect HIV-infected T lymphoblastoid cells (MT4 cells) from cell-death mediated by the infection. The endpoint of the assay was calculated as the concentration of compound at which the cell viability of the culture was preserved by 50% (xe2x80x9850% inhibitory concentrationxe2x80x99, IC50). The cell viability of a culture was determined by the uptake of soluble, yellow 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and its reduction to a purple insoluble formazan salt. After solubilization, spectrophotometric methods were employed to measure the amount of formazan product.
MT4 cells were prepared to be in logarithmic-phase growth and a total of 2xc3x97106 cells infected with either the wild type or site directed mutant clones of HIV-HXB2 at a multiplicity of approximately 0.0001 infectious units of virus per cell in a total volume of between 200-500 xcexcl. The cells were incubated with virus for one hour at 37xc2x0 C. then washed in 0.01 M phosphate buffered saline, pH 7.2, and resuspensed in culture medium for incubation in culture with serial dilutions of test compound. The culture medium used was RPMI 1640 without phenol red, supplemented with penicillin, streptomycin, L-glutamine and 10% fetal calf serum (GM10).
Test compounds were prepared as 2 mM solutions in dimethyl sulphoxide (DMSO). Four replicate, serial 2-fold dilutions in GM10 were then prepared and 50 microliter amounts placed in 96-well plates over a final concentration range of 625-1.22 nm. Fifty microliters GM10 and 3.75xc3x97104 infected cells were then added to each well. Control cultures containing no cells (blank), uninfected cells (100% viability; 4 replicates) and infected cells without compound (total virus-mediated cell death; 4 replicates) were also prepared. The cultures were then incubated at 37xc2x0 C. in a humidified atmosphere of 5% CO2 in air for 5 days.
A fresh solution of 5 mg/mL MTT was prepared in 0.01 M phosphate buffered saline, pH 7.2 and 20 xcexcL added to each culture. The cultures were further incubated as before for 2 hours. They were then mixed by pipetting up and down, and 170 microliters of Triton X-100 in acidified isopropanol (10% v/v Triton X-100 in 1:250 mixture of concentrated HCl in isopropanol) were added and the cultures were mixed again by pipetting up and down. When the formazan deposit was fully solubilized by further mixing, the absorbance (OD) of the cultures was measured at 540 nm and 690 nm wavelength (690 nm readings were used as blanks for artefacts between wells). The percent protection for each treated culture was then calculated from the equation:
OD drug-treated cultures)xe2x88x92(OD untreated virus control cultures) % Protection=100%
(OD uninfected cultures)xe2x88x92(OD untreated virus control cultures)
The IC50 was then obtained from graph plots of percentage protection versus log10 drug concentration.
The IC50 of the compounds of the present invention is as a rule in the range of 1 nM to 10,000 nM, preferably in the range of 1 nM to 60 nM.
Some representative activity data is given in table 9 below.
Enzyme inhibitory IC50s have been rounded up to 1 decimal point, antiviral IC50s have been rounded up to nearest whole number.
The compounds of the present invention, as well as their pharmaceutically usable acid addition salts, can be used as medicaments, e.g. in the form of pharmaceutical preparations. The pharmaceutical preparations can be administered orally, e.g. in the form of tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, emulsions or suspensions. The administration can, however, also be effected rectally, e.g. in the form of suppositories, or parenterally, e.g. in the form of injection solutions.
The compounds of the present invention and their pharmaceutically usable acid addition salts can be processed with pharmaceutically inert, inorganic or organic excipients for the production of tablets, coated tablets, dragees and hard gelatin capsules. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts etc. can be used as such excipients e.g. for tablets, dragxc3xa9es and hard gelatine capsules.
Suitable excipients for soft gelatine capsules are e.g. vegetable oils, waxes, fats, semi-solid and liquid polyols etc.
Suitable excipients for the manufacture of solutions and syrups are e.g. water, polyols, saccharose, invert sugar, glucose etc.
Suitable excipients for injection solutions are e.g. water, alcohols, polyols, glycerol, vegetable oils etc.
Suitable excipients for suppositories are e.g. natural or hardened oils, waxes, fats, semi-liquid or liquid polyols etc.
Moreover, the pharmaceutical preparations can contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.
The dosage can vary within wide limits and will, of course, be fitted to the individual requirements in each particular case. In general, in the case of oral administration a daily dosage of about 10 to 2500 mg per person of a compound of formula I should be appropriate, although the above upper limit can also be exceeded when necessary.
The daily dosage can be administered as a single dosage or in divided dosages. The treatment may be in conjunction with the administration of one or more additional therapeutically active substance(s), and such administration may be concurrent or sequential with respect to that of the compounds of formula I. Thus, concurrent administration, as used herein, includes administration of the agents in conjunction or combination, together, or before or after each other.
Mass spectra were recorded under electrospray ionization conditions on one of the following instruments:
THERMOQUEST SSQ 7000 [Solvent 0.085% TFA in 90% acetonitrile/water; flow rate 100 microliters/minute; capillary 250xc2x0 C.; spray voltage 5KV; sheath gas 80 psi], or LC-MS system (liquid chromatograph coupled to mass spectrum) THERMOQUEST 7000 ELECTROSPRAY or MICROMASS PLATFORM ELECTROSPRAY [gradient of 0.1% TFA in water to 0.085% TFA in acetonitrile]
With regard to the starting materials that are known compounds some of these may be purchased from commercial suppliers. Other known starting materials and their analogues can be prepared by methods well known in the art. Examples of the compounds available from commercial suppliers, and citations to the synthesis of other compounds and their analogues are provided in the following.